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Prague, Czech Republic

Slouf M.,Czech Institute of Macromolecular Chemical | Pavlova E.,Czech Institute of Macromolecular Chemical | Bhardwaj M.,Czech Institute of Macromolecular Chemical | Plestil J.,Czech Institute of Macromolecular Chemical | And 3 more authors.
Materials Letters

Stable Pd nanoparticles (PdNP) with a tunable size (3-15 nm) were synthesized by controlled chemical reduction of PdCl2 with sodium citrate in water. The morphology of PdNP was characterized by transmission electron microscopy, while their stability in solution was verified by quasi-elastic light scattering and small-angle X-ray scattering. Intensive stirring of reacting mixture played a vital role in achieving reproducible particle sizes. Controlled changes of pH and initial concentrations were employed in fine-tuning particle size distributions. Finally, 10 nm PdNP were conjugated with goat anti-mouse IgG antibody as proved by electrophoresis (SDS-PAGE) and used for ultrastructural immunolabeling, which confirmed suitability of PdNP for multiple immunolabeling in biomedicine. © 2011 Elsevier B.V. All rights reserved. Source

Stechova K.,Charles University | Halbhuber Z.,Central European Biosystems | Hubackova M.,Charles University | Kayserova J.,Charles University | And 11 more authors.
European Journal of Human Genetics

Type 1 diabetes (T1D) is an autoimmune disease characterized by the lack of insulin due to an autoimmune destruction of pancreatic beta cells. Here, we report a unique case of a family with naturally conceived quadruplets in which T1D was diagnosed in two quadruplets simultaneously. At the same time, the third quadruplet was diagnosed with the pre-diabetic stage. Remarkably, all four quadruplets were positive for anti-islet cell antibodies, GAD65 and IA-A2. Monozygotic status of the quadruplets was confirmed by testing 14 different short tandem repeat polymorphisms. Serological examination confirmed that all quadruplets and their father suffered from a recent enteroviral infection of EV68-71 serotype. To assess the nature of the molecular pathological processes contributing to the development of diabetes, immunocompetent cells isolated from all family members were characterized by gene expression arrays, immune-cell enumerations and cytokine-production assays. The microarray data provided evidence that viral infection, and IL-27 and IL-9 cytokine signalling contributed to the onset of T1D in two of the quadruplets. The propensity of stimulated immunocompetent cells from non-diabetic members of the family to secrete high level of IFN-α further corroborates this conclusion. The number of T regulatory cells as well as plasmacytoid and/or myeloid dendritic cells was found diminished in all family members. Thus, this unique family is a prime example for the support of the so-called fertile-field hypothesis proposing that genetic predisposition to anti-islet autoimmunity is fertilized and precipitated by a viral infection leading to a fully blown T1D. © 2012 Macmillan Publishers Limited All rights reserved. Source

Korabecna M.,Charles University | Geryk J.,Central European Biosystems | Hora M.,Charles University | Steiner P.,Bioptic Laboratory | And 2 more authors.

Tubulocystic renal cell carcinoma (TRCC) represents a rare tumor with incidence lower than 1 % of all renal carcinomas. This study was undertaken to contribute to characterization of molecular signatures associated with TRCC and to compare them with the features of papillary renal cell carcinoma (PRCC) at the level of genome wide methylation analysis. We performed methylated DNA immunoprecipitation (MeDIP) coupled with microarray analysis (Roche NimbleGen). Using the CHARM package, we compared the levels of gene methylation between paired samples of tumors and control renal tissues of each examined individual. We found significant global demethylation in all tumor samples in comparison with adjacent kidney tissues of normal histological appearance but no significant differences in gene methylation between the both compared tumor entities. Therefore we focused on characterization of differentially methylated regions between both tumors and control tissues. We found 42 differentially methylated genes. Hypermethylated genes for protocadherins (PCDHG) and genes coding for products associated with functions of plasma membrane were evaluated as significantly overrepresented among hypermethylated genes detected in both types of renal cell carcinomas. In our pilot study, we provide the first evidence that identical features in the process of carcinogenesis leading to TRCC and/or to PRCC may be found at the gene methylation level. © 2016, Cancer Research Institute Slovak Acad. of Sciences. All rights reserved. Source

Vanecek T.,Bioptical Laboratory | Halbhuber Z.,Central European Biosystems | Kacerovska D.,Bioptical Laboratory | Kacerovska D.,Charles University | And 6 more authors.
American Journal of Dermatopathology

Brooke-Spiegler syndrome (BSS) and its phenotypic variants, multiple familial trichoepithelioma (MFT) and familial cylindromatosis, are rare autosomal dominant hereditary diseases. They are characterized by the presence of multiple adnexal tumors, especially cylindromas, spiradenomas, spiradenocylindromas, and trichoepitheliomas. Implicated in the pathogenesis of the disease is the gene CYLD, which is localized on the long arm of chromosome 16. This gene encodes an evolutionarily conserved protein belonging to the deubiquitinating enzymes family, which plays a key role in many signaling pathways, especially in NF-kB, JNK, and Wnt. Less than 90 germline mutations of CYLD have been identified in patients with BSS/MFT. These mutations are mostly small alterations in the coding sequence and at exon-intron junction sites. One patient with an intronic mutation and another with a large CYLD deletion have also been recorded. In this study, the authors have analyzed a cohort of 14 patients with BSS/MFT from 13 failies for large genome rearrangements by array comparative genome hybridization followed by confirmatory sequencing. We identified 2 large deletions, namely c.-34111-297858del378779 and c.914-6398-1769del13642ins20 in patients with MFT and BSS, respectively. All other analyzable patients did not reveal any copy number alteration. It is concluded that the large rearrangements are relatively rare in patients without a germline CYLD mutation demonstrable by conventional sequencing. The pathogenetic mechanisms in patients with BSS/MFT lacking germline sequence alterations or large rearrangements in the CYLD gene remain to be clarified. © 2014 Lippincott Williams & Wilkins. Source

Vcelakova J.,Charles University | Blatny R.,Central European Biosystems | Halbhuber Z.,Central European Biosystems | Kolar M.,Academy of Sciences of the Czech Republic | And 9 more authors.
Journal of Diabetes Research

Type 1 Diabetes (T1D) is considered to be a T-helper- (Th-) 1 autoimmune disease; however, T1D pathogenesis likely involves many factors, and sufficient tools for autoreactive T cell detection for the study of this disease are currently lacking. In this study, using gene expression microarrays, we analysed the effect of diabetes-associated autoantigens on peripheral blood mononuclear cells (PBMCs) with the purpose of identifying (pre)diabetes-associated cell processes. Twelve patients with recent onset T1D, 18 first-degree relatives of the TD1 patients (DRL; 9/18 autoantibody positive), and 13 healthy controls (DV) were tested. PBMCs from these individuals were stimulated with a cocktail of diabetes-associated autoantigens (proinsulin, IA-2, and GAD65-derived peptides). After 72 hours, gene expression was evaluated by high-density gene microarray. The greatest number of functional differences was observed between relatives and controls (69 pathways), from which 15% of the pathways belonged to "immune response-related" processes. In the T1D versus controls comparison, more pathways (24%) were classified as "immune response-related." Important pathways that were identified using data from the T1D versus controls comparison were pathways involving antigen presentation by MHCII, the activation of Th17 and Th22 responses, and cytoskeleton rearrangement-related processes. Genes involved in Th17 and TGF-beta cascades may represent novel, promising (pre)diabetes biomarkers. © 2013 Jana Vcelakova et al. Source

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